WO2024120317A1

WO2024120317A1 - Supramolecular tranexamic acid-mandelic acid ionic salt as well as preparation method therefor and use thereof

Info

Publication number: WO2024120317A1
Application number: PCT/CN2023/135958
Authority: WO
Inventors: 王振元; 张嘉恒; 李诺; 刘俊光; 周丽; 姚远志; 苏晶
Original assignee: 深圳杉海创新技术有限公司
Priority date: 2022-12-07
Filing date: 2023-12-01
Publication date: 2024-06-13
Also published as: CN116217379A; CN116217379B

Abstract

Provided are a supramolecular tranexamic acid-mandelic acid ionic salt as well as a preparation method therefor and a use thereof, relating to the technical field of pharmaceutical and cosmetic compounds. The supramolecular tranexamic acid-mandelic acid ionic salt has a structural formula as shown in formula I. Tranexamic acid and mandelic acid undergo reaction and bonding to form a supramolecular salt. The skin permeability of mandelic acid can be effectively improved and less irritation is caused to the skin while the efficacy of mandelic acid is well maintained.

Description

Supramolecular tranexamic acid mandelic acid ion salt and its preparation method and application

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of Chinese patent application No. 202211560438.6 filed with the Chinese Patent Office on December 7, 2022, and entitled “Supramolecular tranexamic acid mandelic acid ion salt and its preparation method and application”, the entire contents of which are incorporated by reference into this application.

Technical Field

The present application relates to the technical field of compounds for medicine and cosmetics, and in particular to a supramolecular tranexamic acid mandelate ion salt and a preparation method and application thereof.

Background technique

Mandelic acid (MA), also known as mandelic acid and phenylethylic acid, has a chemical structure of α-hydroxyphenylacetic acid. It is an important chiral drug intermediate and fine chemical product. For example, it is the raw material for the synthesis of drugs such as the vasodilator cyclomandelate, the anti-inflammatory drug for urinary tract infection, and the antispasmodic drug benzyl mandelate.

Transdermal administration is currently a common way to use mandelic acid products. For example, transdermal administration can solve inflammation problems. However, conventional mandelic acid topical preparations are difficult to penetrate the stratum corneum barrier, resulting in low bioavailability of mandelic acid and limited application.

Application Contents

The purpose of the present application is to provide a supramolecular tranexamic acid mandelic acid ion salt and its preparation method and application, which can effectively improve the skin permeability of mandelic acid while better maintaining the efficacy of mandelic acid and has low irritation to the skin.

The embodiment of the present application is implemented as follows:

In a first aspect, the present invention provides a supramolecular tranexamic acid mandelate ion salt having a structural formula as shown in Formula I:

In a second aspect, an embodiment of the present application provides a method for preparing a supramolecular tranexamic acid mandelic acid ion salt as provided in the embodiment of the first aspect, which comprises reacting tranexamic acid and mandelic acid to obtain a supramolecular tranexamic acid mandelic acid ion salt as shown in Formula I.

In a third aspect, an embodiment of the present application provides a use of a supramolecular tranexamic acid mandelate ion salt as provided in an embodiment of the first aspect as a raw material in the preparation of medicines or cosmetics.

The supramolecular tranexamic acid mandelic acid ion salt and its preparation method and application provided in the embodiments of the present application have the following beneficial effects: In the present application, tranexamic acid and mandelic acid are used as precursors, and a supramolecular tranexamic acid mandelic acid ion salt as shown in Formula I is obtained by ionization salt-forming reaction. The supramolecular tranexamic acid mandelic acid ion salt can better maintain the efficacy of mandelic acid, such as antioxidant properties, inhibition of melanocyte activity, and inhibition of tyrosinase activity; at the same time, it can effectively improve the skin permeability of mandelic acid and improve the bioavailability of mandelic acid; moreover, it has low irritation to the skin, which enhances the application effect.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

FIG1 is a schematic flow diagram of a method for preparing a supramolecular tranexamic acid mandelate ion salt provided in an embodiment of the present application;

FIG2 is a thermogravimetric analysis curve of the supramolecular tranexamic acid mandelic acid ion salt, tranexamic acid monomer, and mandelic acid monomer in Example 1 of the present application;

FIG3 is a hydrogen NMR spectrum of the supramolecular tranexamic acid mandelate ion salt in Example 1 of the present application;

FIG4 is a schematic diagram of the molecular structure of the supramolecular tranexamic acid mandelate ion salt crystal in Example 5 of the present application;

FIG5 is a statistical diagram of the penetration efficiency of Test Example 1 of the present application;

FIG6 is a line graph of DPPH free radical scavenging rate of Test Example 4 of the present application;

FIG. 7 is a line graph of the ABTS ⁺ · removal rate of Test Example 4 of the present application.

Detailed ways

The technical solution of the present application will be described in detail below in conjunction with the specific implementation methods. If the specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

In a first aspect, the embodiments of the present application provide a supramolecular tranexamic acid mandelate ion salt having a structural formula as shown in Formula I;

In some possible embodiments, the supramolecular tranexamic acid mandelic acid ion salt comprises a tranexamic acid structure and a mandelic acid structure in a molar ratio (i.e., a ratio of the amount of substance) of 1:5 to 5:1. In this embodiment, the tranexamic acid structure and the mandelic acid structure have a suitable ratio of the amount of substance, so that the supramolecular tranexamic acid mandelic acid ion salt can have a better penetration effect.

As an example, the molar ratio of the tranexamic acid structure to the mandelic acid structure is, for example but not limited to, any one of 1:5, 1:4, 1:3, 1:2, 1:1, 2:5, 2:3, 2:1, 3:5, 3:4, 3:2, 3:1, 4:5, 4:3, 4:1, 5:4, 5:3, 5:2 and 5:1, or a range between any two of them.

Referring to Figure 1, as an example, the step of reacting tranexamic acid and mandelic acid to obtain a supramolecular tranexamic acid mandelic acid ion salt as shown in Formula I comprises the following operations: in a protective gas atmosphere, tranexamic acid and mandelic acid are added to an organic solvent, reacted for a predetermined time, and then ultrasonicated and stirred to obtain a supramolecular tranexamic acid mandelic acid ion salt solution. The supramolecular tranexamic acid mandelic acid ion salt solution is crystallized, filtered and dried to obtain a supramolecular tranexamic acid mandelic acid ion salt. Wherein, the protective gas atmosphere refers to an anti-oxidative protective atmosphere, which is, for example, an atmosphere of one or more inert gases including helium, argon, nitrogen, carbon dioxide, etc.

The usage ratio of tranexamic acid and mandelic acid can refer to the molar ratio of tranexamic acid structure and mandelic acid structure in the supramolecular tranexamic acid mandelic acid ion salt, that is, as an example, the molar ratio of tranexamic acid and mandelic acid is 1:5 to 5:1.

The type of organic solvent is not limited, as long as it can achieve good dissolution and dispersion of tranexamic acid and mandelic acid. As an example, the organic solvent includes one or more of acetonitrile, ethanol and methanol.

In order to allow tranexamic acid and mandelic acid to undergo a more complete ionization and salt-forming reaction, optionally, the predetermined time is 12 h to 48 h. The predetermined time for the ionization and salt-forming reaction is, for example but not limited to, any one of 12 h, 18 h, 24 h, 30 h, 36 h, 42 h and 48 h, or a range between any two of them.

In order to achieve a better ultrasonic effect, optionally, during the ultrasonic process, at least one of the following conditions (a1) to (a5) is met: (a1) The temperature of the ultrasonic field is 50°C to 90°C, for example but not limited to any one of 50°C, 60°C, 70°C, 80°C and 90°C or a range between any two of them. (a2) The ultrasonic frequency is 20kHz to 60kHz, for example but not limited to any one of 20kHz, 30kHz, 40kHz, 50kHz and 60kHz or a range between any two of them. (a3) The ultrasonic power is 700W to 6000W, for example but not limited to any one of 700W, 1000W, 1500W, 2000W, 2500W, 3000W, 3500W, 4000W, 4500W, 5000W, 5500W and 6000W or a range between any two of them. (a4) The ultrasound time is 6 hours to 12 hours, for example but not limited to any one of 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours and 12 hours, or a range between any two of them. (a5) Ultrasound is performed for 2 seconds to 10 seconds every 1 second to 5 seconds, wherein the ultrasound interval time is for example but not limited to 1 second, 2 seconds, 3 seconds, The ultrasonic time between two intervals is, for example but not limited to, any point value among 2s, 3s, 4s, 5s, 6s, 7s, 8s, 9s and 10s, or a range value between any two of them.

In order to achieve a better stirring effect, optionally, during the stirring process, the following conditions (b1) and/or (b2) are met.

(b1) The stirring rate is 30 rad/min to 250 rad/min, for example but not limited to any one of 30 rad/min, 50 rad/min, 100 rad/min, 150 rad/min, 200 rad/min and 250 rad/min, or a range between any two of them. (b2) The stirring time is 12 h to 48 h, for example but not limited to any one of 12 h, 18 h, 24 h, 30 h, 36 h, 42 h and 48 h, or a range between any two of them.

In the present application, the crystallization method is not limited, as long as the supramolecular tranexamic acid mandelate ion salt obtained by the reaction can be effectively crystallized and separated from the salt solution. Optionally, during the crystallization process, the following conditions (c1) and/or (c2) are met.

(c1) performing concentrated crystallization under vacuum conditions. (c2) performing cooling crystallization under a temperature condition of 5°C to 15°C, wherein the temperature of cooling crystallization is, for example but not limited to, any one of 5°C, 8°C, 10°C, 12°C and 15°C or a range between any two of them.

In order to achieve efficient drying without destroying the supramolecular tranexamic acid mandelic acid ion salt, the drying temperature is optionally 50°C to 90°C, for example but not limited to any one of 50°C, 60°C, 70°C, 80°C and 90°C or a range between any two thereof. The drying time depends on the degree of drying, and can be optionally 36h to 60h, for example but not limited to any one of 36h, 42h, 48h, 54h and 60h or a range between any two thereof.

As an example, medicines and cosmetics are each, for example but not limited to, products for achieving one or more of the following target functions, including, for example: anti-oxidation, inhibition of melanocyte activity, inhibition of tyrosinase activity, DPPH·free radical scavenging, ABTS ⁺ ·scavenging, and anti-inflammatory.

The technical solution of the present application will be described below in conjunction with specific embodiments and test examples.

1. Examples and Comparative Examples

Example 1

A supramolecular tranexamic acid mandelate ion salt, the preparation method of which is as follows:

In an inert gas atmosphere, 0.10 mol of tranexamic acid and 0.10 mol of mandelic acid were added to acetonitrile, and the reaction time was 24 hours. Under the condition of 75°C, the ultrasonic frequency was 40kHz, the ultrasonic power was 2000W, the ultrasonic time was 12 hours, and the interval time was 10 seconds every 3 seconds. The stirring time was 24 hours and the stirring rate was 60 rad/min, and a supramolecular tranexamic acid mandelic acid ion salt solution was obtained. Under vacuum conditions, the obtained supramolecular tranexamic acid mandelic acid ion salt solution was concentrated and crystallized; it was dried in a vacuum drying oven for 48 hours at a drying temperature of 60°C to obtain a supramolecular tranexamic acid mandelic acid ion salt.

Example 2

A supramolecular tranexamic acid mandelic acid ion salt, the preparation method of which is as follows: in an inert gas atmosphere, 0.10 mol of tranexamic acid and 0.10 mol of mandelic acid are added to acetonitrile, the reaction time is 24 hours; under the condition of 50°C, the ultrasonic frequency is 20 kHz, the ultrasonic power is 700 W, the ultrasonic time is 6 hours, the intermittent time is 10 seconds every 3 seconds of ultrasonication; the stirring time is 24 hours, and the stirring rate is 60 rad/min, to obtain a supramolecular tranexamic acid mandelic acid ion salt solution. Under vacuum conditions, the obtained supramolecular tranexamic acid mandelic acid ion salt solution is concentrated and crystallized; and it is dried in a vacuum drying oven for 48 hours at a drying temperature of 60°C to obtain a supramolecular tranexamic acid mandelic acid ion salt.

Example 3

A supramolecular tranexamic acid mandelic acid ion salt, the preparation method of which is as follows: in an inert gas atmosphere, 0.10 mol of tranexamic acid and 0.10 mol of mandelic acid are added to acetonitrile, the reaction time is 24 hours; under the condition of 90°C, the ultrasonic frequency is 60kHz, the ultrasonic power is 6000W, the ultrasonic time is 12 hours, the intermittent time is 10 seconds every 3 seconds of ultrasonication; the stirring time is 24 hours, and the stirring rate is 60rad/min, to obtain a supramolecular tranexamic acid mandelic acid ion salt solution. Under vacuum conditions, the obtained supramolecular tranexamic acid mandelic acid ion salt solution is concentrated and crystallized; and it is dried in a vacuum drying oven for 48 hours at a drying temperature of 60°C to obtain a supramolecular tranexamic acid mandelic acid ion salt.

Example 4

A supramolecular tranexamic acid-mandelic acid ion salt, which is different from Example 1 in that the amount of tranexamic acid is 6 mol, and the amount of mandelic acid is 1 mol.

Example 5

A supramolecular tranexamic acid mandelate ion salt crystal, the preparation method of which is as follows:

In an inert gas atmosphere, 0.10 mol of tranexamic acid and 0.10 mol of mandelic acid were placed in acetonitrile; at 75°C, the ultrasonic frequency was 40 kHz, the ultrasonic power was 2000 W, the ultrasonic time was 12 h, the intermittent time was 10 s every 3 s; the stirring time was 24 h, and the stirring rate was 60 rad/min, to obtain a supramolecular tranexamic acid mandelic acid ion salt solution. Under low temperature conditions, the obtained supramolecular tranexamic acid mandelic acid ion salt solution was crystallized at a crystallization temperature of 10°C, and the supramolecular tranexamic acid mandelic acid crystals were obtained after filtration.

Comparative Example 1

A supramolecular ion salt, which is different from Example 1 in that mandelic acid is replaced with citric acid of the same substance amount to prepare a supramolecular tranexamic acid citrate ion salt.

2. Material performance test

(1) The thermal decomposition behaviors of supramolecular tranexamic acid-mandelic acid ion salt, tranexamic acid monomer, and mandelic acid monomer at a heating rate of 5.0 K/min were studied using thermogravimetric technology. The results are shown in Figure 2.

As shown in Figure 2, the supramolecular tranexamic acid mandelic acid ion salt begins to undergo thermal cracking at around 190°C, indicating that it is stable at room temperature. The thermal cracking of tranexamic acid and mandelic acid occurs at around 77°C and 135°C, respectively, that is, the melting point of the supramolecular tranexamic acid mandelic acid ion salt is lower than that of the tranexamic acid monomer, indicating that the ion salt is effectively formed.

(2) As shown in Figure 3, the H NMR spectrum data of the supramolecular tranexamic acid mandelate ion salt prepared in this embodiment are: 1H NMR (600MHz, D2O) δ7.47-7.27 (m, 5H), 5.00 (s, 1H), 2.81 (d, J = 7.1 Hz, 2H), 2.25 (tt, J = 12.3, 3.5 Hz, 1H), 1.99-1.92 (m, 2H), 1.80 (dd, J = 9.5, 4.1 Hz, 2H), 1.69-1.51 (m, 1H), 1.36 (qd, J = 13.0, 3.3 Hz, 2H), 1.02 (qd, J = 12.9, 3.4 Hz, 2H).

(3) As shown in FIG4 , the supramolecular tranexamic acid-mandelic acid crystals obtained in Example 5 were subjected to X-ray single crystal diffraction test. The specific test parameters were: SuperNova, Dual, Cu at zero, AtlasS2 diffractometer, temperature of 149.99(10)K, and structural analysis using Olex2 and ShelXL.

The X-ray single crystal diffraction test results of the supramolecular tranexamic acid-mandelic acid crystals are shown in Table 1.

Table 1. Single crystal data of supramolecular tranexamic acid mandelate ion salt

Atomic coordinates (×10 ⁴ ) and isotropic atomic displacement parameters of supramolecular tranexamic acid-mandelic acid The analytical data are shown in Table 2, where U(eq) is defined as one third of the trace of the orthogonal U _ij tensor.

Table 2. Atomic coordinates and isotropic atomic displacement parameters of supramolecular tranexamic acid mandelate ion salt

The anisotropic atomic displacement parameter analysis data of supramolecular tranexamic acid mandelate ion salt are shown in Table 3, wherein the anisotropic atomic displacement factor power is expressed as: -2π ² [h ² a* ² U ₁₁ +2hka*b*U ₁₂ +…].

Table 3. Anisotropic atomic displacement parameters of supramolecular tranexamic acid mandelate ion salt

The bond length analysis data of the supramolecular tranexamic acid mandelate ion salt are shown in Table 4.

Table 4. Bond lengths of supramolecular tranexamic acid mandelate ion salt

The analysis data of the chemical bond angles (°) of the supramolecular tranexamic acid mandelate ion salt are shown in Table 5.

Table 5. Bond angles of various chemical bonds of supramolecular tranexamic acid mandelate ion salt

The hydrogen bond analysis data of the supramolecular tranexamic acid mandelate ion salt are shown in Table 6.

Table 6. Hydrogen bonds of supramolecular tranexamic acid mandelate ion salt

The analysis data of the torsion angles (°) of each chemical bond of the supramolecular tranexamic acid mandelate ion salt are shown in Table 7.

Table 7. Chemical bond torsion angles of supramolecular tranexamic acid mandelate ion salt

Hydrogen Atom Coordinates of Supramolecular Tranexamic Acid Mandelic Acid Ion Salt and the isotropic atomic displacement parameters The analysis data are shown in Table 8.

Table 8. Hydrogen atom coordinates and isotropic atomic displacement parameters of supramolecular tranexamic acid mandelate ion salt

3. Application Test Examples

Test Example 1

The supramolecular tranexamic acid mandelic acid ion salt prepared in Example 1 was prepared into a 10wt% supramolecular tranexamic acid mandelic acid solution, and a 4.92wt% mandelic acid solution was prepared according to the amount of mandelic acid and the like for comparison. The above solutions were tested for transdermal effect. The specific test method was as follows: 1. The back skin of GF Kunming mice was used, the subcutaneous fat layer and connective tissue were carefully peeled off, and rinsed with physiological saline. Place in physiological saline for later use. II. Perform transdermal experiments using the Franz cell method. The exposed mouse skin area in the diffusion cell of the Franz diffusion device is 1.13 ^cm2 , and the volume of the receiving chamber is 15 mL. III. Take 1.0 mL of the supramolecular tranexamic acid-mandelic acid solution and an equal amount of mandelic acid solution respectively, as the test solution, and place them on the exposed skin surface in the diffusion cell. Add 15 mL of physiological saline receiving solution to the receiving cell, place it in a 32±0.5℃ constant temperature water bath, and stir at a speed of 350 rad/min. IV. Test of subcutaneous permeability: Take 1 mL of receiving solution at different time points, and conduct 3 replicates at each time point. Immediately after sampling, add 1 mL of receiving solution to the receiving chamber, filter through a 0.22μm microporous filter membrane, and perform HPLC detection. Calculate the cumulative permeability of mandelic acid through the skin. Formula (1) is as follows:
Q＝Cn×V+∑Ci×V ₀ (i＝1…n-1).............................................Formula (2).

Wherein, Q: cumulative permeation amount, μg; V: volume of receiving solution in the receiving chamber, 15 mL; V ₀ : volume of each sampling, 1.0 mL; Ci: drug concentration in the receiving solution from the 1st to the n-1st sampling; Cn: sample concentration measured at the nth sampling point.

Figure 5 is a statistical chart of the penetration effect. It can be seen from Figure 5 that the penetration amount of mandelic acid in the 10% supramolecular tranexamic acid-mandelic acid solution is higher than that of the mandelic acid solution with the same amount of substance in each time period, and the cumulative penetration amount of mandelic acid at the 24th hour is 1.89 times that of the mandelic acid solution with the same amount of substance.

The supramolecular ionic salts of Examples 1 to 4 and Comparative Example 1 were respectively prepared into 10 wt% supramolecular ionic salt solutions, which were named supramolecular tranexamic acid mandelic acid solution 1, supramolecular tranexamic acid mandelic acid solution 2, supramolecular tranexamic acid mandelic acid solution 3, supramolecular tranexamic acid mandelic acid solution 4 and supramolecular tranexamic acid citric acid solution 1, and transdermal effect tests were carried out according to the above method. The test results are shown in Table 9.

Table 9. Penetration effect statistics

From the comparison between Examples 1 to 4 and Comparative Example 1, and between Example 1 and Example 4, it can be seen that when the molar ratio of tranexamic acid to mandelic acid is in the range of 1:5 to 5:1, the prepared supramolecular tranexamic acid-mandelic acid has better penetration effect, indicating that a specific ratio of tranexamic acid and mandelic acid can play a role in improving the penetration effect, and this effect is stronger than that of ionic salts made from other ligands, such as supramolecular tranexamic acid citrate ion salt.

Test Example 2

According to T/SHRH027-2019 "In vitro test of B16 cell melanin synthesis inhibition experiment" and T-SHRH015-2018 "Cosmetics-Tyrosinase activity inhibition test method", based on melanoma cells (B16) cytotoxicity detection, the maximum safe dosage of the supramolecular tranexamic acid mandelic acid ion salt of Example 1 and the supramolecular tranexamic acid citrate ion salt of Comparative Example 1 on melanoma cells was determined, and the cellular melanin content, tyrosinase activity and tyrosinase inhibition rate were tested within the safe concentration.

The results of the cytotoxicity test are shown in Tables 10 and 11 below.

Table 10. Cytotoxicity test results of supramolecular tranexamic acid mandelic acid ion salt

Table 11. Cytotoxicity test results of supramolecular tranexamic acid citrate ion salt

According to the cytotoxicity results, both samples showed no melanoma cell toxicity within the concentration range of 0.002% (m/V).

The results of the cell melanin synthesis inhibition test are as follows:

Table 12. Analysis of the results of cell melanin synthesis inhibition rate

Note: When the t-test method was used for statistical analysis, the significance of the PC group and the sample group compared with the BC group was indicated by *, p-value < 0.05 was indicated by *, and p-value < 0.01 was indicated by **.

The results of cell tyrosinase activity inhibition are as follows:

Table 13. Cellular tyrosinase activity inhibition test results

The results of in vitro tyrosinase inhibition assay are as follows:

Table 14. Analysis of in vitro tyrosinase inhibition test results

In Tables 12 to 14, the BC group is the blank control group, and the PC group is the positive control group.

The results of the cell melanin synthesis inhibition test are shown in Table 12. After the cells were treated with 0.0006%, 0.0012%, and 0.002% (m/V) supramolecular tranexamic acid mandelic acid ion salt and supramolecular tranexamic acid citrate ion salt, the melanin synthesis inhibition rate increased with the increase of sample concentration, and there was a statistical difference compared with the blank control (p<0.05), indicating that it has an inhibitory effect on the cell melanin synthesis ability, and the supramolecular tranexamic acid mandelic acid ion salt has a higher melanin synthesis inhibition rate than the supramolecular tranexamic acid citrate ion salt.

The results of the cell tyrosinase activity inhibition test are shown in Table 13. After the cells were treated with 0.0006%, 0.0012%, and 0.002% (m/V) supramolecular tranexamic acid mandelic acid ion salt and supramolecular tranexamic acid citrate ion salt, the cell tyrosinase activity inhibition rate increased with the increase of sample concentration, and there was a statistical difference compared with the blank control (p<0.05), indicating that they had an inhibitory effect on the cell tyrosinase activity, and the supramolecular tranexamic acid mandelic acid ion salt had a higher inhibition rate on the cell tyrosinase activity than the supramolecular tranexamic acid citrate ion salt.

As shown in Table 14, the inhibition rate of supramolecular tranexamic acid mandelic acid ion salt on tyrosinase at a concentration of 1.25-10% (m/V) was above 80%, which was statistically different from the blank control group (p<0.01), indicating that it had an inhibitory effect on tyrosinase activity; and the inhibition rate of supramolecular tranexamic acid citrate ion salt on tyrosinase at a concentration of 1.25-10% (m/V) was above 60%, indicating that the inhibitory effect of supramolecular tranexamic acid mandelic acid ion salt on tyrosinase activity was higher than that of supramolecular tranexamic acid citrate ion salt.

Test Example 3

The anti-inflammatory effect of the supramolecular tranexamic acid mandelate ion salt prepared in Example 1 was detected by the following method:

TPA-induced mouse ear swelling model and administration method: SPF male KM mice weighing 20g-23g were placed in a temperature of 25°C and a relative humidity of 40%-70% for 24h (12h each under the conditions of light and dark alternation during the day and night), and were given sufficient food and water to allow them to eat freely. The mice were randomly divided into 6 groups, a blank group (BC), a negative control group (NC), a positive control group (PC) and 2 drug groups. The supramolecular tranexamic acid mandelic acid ion salt (0.8mg/ear) and mandelic acid (0.39mg/ear) were evenly applied to the inside and outside of the right ear of the mice in the drug group. After 15 minutes, phorbol ester (TPA) was evenly applied to the inside and outside of the right ear of the mice at 2.0μg/ear to induce inflammation. After 6 hours, the mice were immediately killed by cervical dislocation, the left and right ears of the mice were cut off and weighed, the ear tissue of the mice was crushed in liquid nitrogen, transferred to a 4mL EP tube, 1mL T-PER tissue protein extraction reagent was added, and the mixture was fully suspended for about 30 minutes, and then the ear tissue was fully lysed by intermittent 10s at 20Hz and ultrasonic for 2min, and then centrifuged at 4℃ and 13000r/min for 20min. The supernatant was taken and the content of inflammatory factors IL-1α, TNF-α, and MIP-2 in the ear tissue was determined by ELISA.

The results of the effects of supramolecular tranexamic acid mandelic acid ion salt and mandelic acid monomer on IL-1α, TNF-α, and MIP-2 inflammatory factors in mouse ear tissue are as follows:

Table 15. Summary of IL-1α test results

Table 16. Summary of TNF-α test results

Table 17. Summary of MIP-2 test results

The BC group is the blank control group, the NC group is the negative control group, and the PC group is the positive control group. As can be seen from Tables 15 to 17, the IL-1α, TNF-α and MIP-2 contents of the negative control group increased significantly compared with the blank group; compared with the negative control group, the IL-1α, TNF-α and MIP-2 contents of the positive control group decreased significantly; indicating that this experiment is effective. Compared with the negative control group, the IL-1α, TNF-α and MIP-2 contents of the supramolecular tranexamic acid mandelic acid ion salt group and the mandelic acid monomer group of the present invention decreased, while the IL-1α, TNF-α and MIP-2 contents of the supramolecular tranexamic acid mandelic acid ion salt group decreased more significantly. Under the condition of the same mandelic acid content, the effect of the supramolecular tranexamic acid mandelic acid ion salt on the decrease of IL-1α, TNF-α and MIP-2 was 1.59 times, 1.65 times and 1.56 times that of the mandelic acid monomer, respectively. This indicates that both supramolecular tranexamic acid mandelic acid ion salt and mandelic acid monomer have anti-inflammatory effects, and the anti-inflammatory effect of supramolecular tranexamic acid mandelic acid ion salt on IL-1α, TNF-α and MIP-2 inflammatory factors is more significant.

Test Example 4

The supramolecular tranexamic acid-mandelic acid ion salt and mandelic acid monomer obtained in Example 1 were prepared into solutions with concentrations of 50 μg/mL, 100 μg/mL, 150 μg/mL, 200 μg/mL, and 250 μg/mL, and the DPPH· free radical scavenging rate and ABTS ⁺ · scavenging rate were measured. The test method is as follows:

Determination of DPPH· free radical scavenging rate: Take 0.2mL of sample solution with different mass concentrations, add 0.8mL of 60μmol/L DPPH· solution (prepared with anhydrous ethanol), mix well and react for 30min (protected from light), measure the absorbance at 517nm wavelength as _A1 , use 70% ethanol solution instead of sample to measure the absorbance as _A0 , use anhydrous ethanol instead of DPPH· solution to measure the absorbance as _A2 , and use VC as the positive control in the experiment. The calculation of DPPH· free radical scavenging rate is shown in formula (2):

ABTS ⁺ · Determination of clearance rate: ABTS solution (7mmol/L) and potassium persulfate solution (2.45mmol/L) were mixed at a ratio of 1:1 (V/V) to prepare ABTS stock solution, and then placed at room temperature for 12h to 16h (protected from light). Dilute with saline buffer, and measure the absorbance A ₀ of the diluent at 734nm wavelength, which should reach 0.70±0.002. Take 50μL of sample solution with different mass concentrations, add 750μL of ABTS determination solution, mix well, react for 6min, and measure the absorbance A ₁ at 734nm wavelength. Use anhydrous ethanol solution instead of ABTS solution to measure the absorbance A ₂ . Use VC as the positive control in the experiment. The calculation of ABTS ⁺ · clearance rate is the same as formula (2).

The results of DPPH· free radical scavenging rate are shown in Figure 6. Supramolecular tranexamic acid mandelic acid ion salt, mandelic acid and VC all have the ability to scavenge DPPH· free radicals. VC has the best scavenging effect, followed by supramolecular tranexamic acid mandelic acid ion salt, and mandelic acid has the worst effect. Moreover, the greater the mass concentration of supramolecular tranexamic acid mandelic acid ion salt, the better the scavenging effect on DPPH· free radicals. This is because the sample can pair with the single electron present in the DPPH· free radical, causing it to be reduced and weakening the color of its purple alcohol solution. Its absorbance is measured at a wavelength of 517nm. The greater the change in absorbance, the stronger its scavenging ability. Therefore, the same concentration of supramolecular tranexamic acid mandelic acid ion salt has a better scavenging effect on DPPH· than mandelic acid monomer.

The results of ABTS ⁺ · clearance rate are shown in Figure 7. The effects of supramolecular tranexamic acid mandelic acid ion salt, mandelic acid and VC in clearing ABTS ⁺ are all significant. VC is higher than supramolecular tranexamic acid mandelic acid ion salt and mandelic acid, followed by supramolecular tranexamic acid mandelic acid ion salt, and mandelic acid has the worst effect. ABTS reacts with oxidants to become ABTS ⁺ · free radicals (blue-green), and the sample will reduce ABTS ⁺ · to colorless ABTS, and its absorbance is measured at a wavelength of 734nm or 405nm. The greater the change in absorbance, the stronger its scavenging ability. Therefore, the same concentration of supramolecular tranexamic acid mandelic acid ion salt has a better scavenging effect on ABTS ⁺ · than mandelic acid monomer.

Test Example 5

The tranexamic acid mandelic acid ion salt obtained in Example 1 was prepared into a 2% aqueous solution, and an equal amount of mandelic acid solution was prepared, and the chicken embryo chorioallantoic membrane test of cosmetic eye irritation/corrosiveness was carried out for comparison. The test method is as follows:

CAM preparation: 9-day-old chick embryos were candled and the eggshell of the air chamber was peeled off with dental serrated curved forceps to expose the white egg membrane. Careful manipulation was performed without damaging the integrity of the egg membrane. A drop of 0.9% sodium chloride solution was dripped with a pipette to moisten the egg membrane. The inner membrane was carefully removed with forceps to ensure that the vascular membrane was not damaged. At this time, the structure of the vascular system was observed again, and its integrity and suitability for the experiment were judged.

Pre-experimental test: Take 2 chicken embryos to check the reactivity of this batch of chicken embryos, and the exposure time is limited to 5 minutes.

Endpoint evaluation method: Take 0.3mL or 0.3g of the test substance and apply it to the CAM, ensuring that at least 50% of the CAM surface is covered by the test substance. After 3 minutes of action, gently rinse the test substance on the CAM with normal saline, and observe the degree of change of each toxic effect about 30 seconds after rinsing.

Reaction evaluation method: Take 0.3mL or 0.3g of the test substance and apply it to the CAM, ensuring that at least 50% of the CAM surface is covered by the test substance. Observe the CAM reaction and record the time of occurrence of each toxic effect within 5 minutes of action.

Six chicken embryos were placed for each sample, and one chicken embryo was set as a negative control and one as a positive control.

Irritation score (IS): For experiments conducted using the reaction time method, the irritation score (IS) was calculated using formula (3), with the result rounded to two decimal places:

Where: secH (bleeding time) - the average time when bleeding begins to occur on the CAM membrane, in seconds (s). secL (vascular melting time) - the average time when vascular melting begins to occur on the CAM membrane, in seconds (s). secC (coagulation time) - the average time when coagulation begins to occur on the CAM membrane, in seconds (s).

As shown in Table 18, the eye irritation of the test substances was classified according to the calculated IS values.

Table 18. Irritation Classification

The test results are as follows:

Table 19. Irritation ratings and results

The results showed that when the concentration of mandelic acid was the same, the supramolecular tranexamic acid-mandelic acid solution was non-irritating, while the mandelic acid solution was slightly irritating. The experiment proved that compared with the mandelic acid monomer, the supramolecular tranexamic acid-mandelic acid ion salt can reduce the irritation of mandelic acid to the skin.

The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application.

Claims

A supramolecular tranexamic acid mandelate ion salt having a structural formula as shown in Formula I;
The supramolecular tranexamic acid-mandelic acid ion salt according to claim 1, wherein it comprises a tranexamic acid structure and a mandelic acid structure in a molar ratio of 1:5 to 5:1.
The supramolecular tranexamic acid mandelic acid ion salt according to claim 1, wherein the single crystal data of the supramolecular tranexamic acid mandelic acid ion salt are as follows:

Chemical formula: C 16 H 23 NO 5 ;

Molecular weight: 309.35;

Temperature: 149.99(10)K;

Crystal system: orthorhombic;

Space group: Pbca;

Unit cell parameters: a: b: c: α: 90°, β: 114.155(11)°, γ: 90°;

volume:

Number of units in the unit cell: 8;

Calculated density: 1.255 g/cm 3 ;

Absorption coefficient: 0.769mm -1 ;

F(0000):1328.0;

Crystal size: 0.13mm×0.11mm×0.09mm

Ray: Cu Kα, λ＝1.54184;

Data collection angle range: 7.416-147.964°;

Index range: -32≤h≤27, -7≤k≤6, -27≤l≤26;

Collected diffraction points: 16532;

Independent diffraction points: 6458, Rint=0.0708, Rsigma=0.0686;

data/restrictions/parameters;6458/0/403;

Goodness of fit for F2 ; 1.062:

Final R values, strength > 2σ: R 1 = 0.0719, wR 2 = 0.1808;

All data R values: R 1 = 0.0944, wR 2 = 0.2072;

Maximum difference peak/well/;
A method for preparing the supramolecular tranexamic acid mandelic acid ion salt as described in any one of claims 1 to 3, wherein it comprises reacting tranexamic acid and mandelic acid to obtain the supramolecular tranexamic acid mandelic acid ion salt as shown in formula I.
The preparation method according to claim 4, wherein it comprises:

In a protective gas atmosphere, the tranexamic acid and the mandelic acid are added to an organic solvent, reacted for a predetermined time, and then subjected to ultrasound and stirring to obtain a supramolecular tranexamic acid mandelic acid ion salt solution;

The supramolecular tranexamic acid mandelic acid ion salt solution is crystallized, filtered and dried to obtain the supramolecular tranexamic acid mandelic acid ion salt.
The preparation method according to claim 5, wherein the predetermined time is 12 hours to 48 hours.
The preparation method according to claim 5, wherein the organic solvent comprises acetonitrile, ethanol and methanol. One or more.
The preparation method according to claim 5, wherein during the ultrasonic process, at least one of the following conditions (a1) to (a5) is satisfied;

(a1) The temperature of the ultrasonic field is 50°C to 90°C;

(a2) Ultrasonic frequency is 20kHz to 60kHz;

(a3) Ultrasonic power is 700W to 6000W;

(a4) Ultrasound time is 6h to 12h;

(a5) Ultrasound for 2s to 10s at intervals of 1s to 5s.
The preparation method according to claim 5, wherein during the stirring process, the following conditions (b1) and/or (b2) are satisfied;

(b1) stirring rate is 30 rad/min to 250 rad/min;

(b2) The stirring time is 12h to 48h.
The preparation method according to claim 5, wherein during the drying process, the drying temperature is 50°C to 90°C.
A use of the supramolecular tranexamic acid mandelate ion salt as claimed in any one of claims 1 to 3 as a raw material in the preparation of medicines or cosmetics.
According to the use of claim 11, the medicine or the cosmetic has the functions of inhibiting melanocyte activity, inhibiting tyrosinase activity, anti-oxidation and anti-inflammatory.